Biomedical subjects

Y Ono

Publications and source records attributed to Y Ono.

At least 289 records · Page 16Linked to original sources

PubMed1998

The effect of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate on CO2 permeability of the red blood cell membrane.

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of 12C18O16O from a red cell suspension as the 18O exchanges between labeled CO2 + HCO3- and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO3- (Pm,HCO-3). To test this technique, we added sufficient 4, 4'-diisothiocyanato-stilbene-2,2'-disulfonate (DIDS) to inhibit all the HCO3-/Cl- transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced Pm,HCO-3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in Pm,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO2 and HCO3-, we found that DIDS inhibited both Pm,HCO-3 and Pm,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO2 exchange.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

The role of PKN in the regulation of alphaB-crystallin expression via heat shock transcription factor 1.

We previously reported that PKN, a fatty acid-activated serine/threonine protein kinase, translocates from the cytosol to the nucleus by stresses such as heat shock, sodium arsenite, and serum starvation. To clarify the role of PKN under heat stress, we examined whether PKN regulates the expression of heat shock proteins. Co-expression of heat shock transcription factor 1 (HSF1) and the catalytically active fragment of PKN induced the accumulation of alphaB-crystallin but not HSP27 and HSP70 in HeLa S3 cells. The expression of the reporter gene for alphaB-crystallin promoter was activated by co-expression of HSF1 and the catalytically active fragment of PKN, and this activation was dependent on the protein kinase activity of PKN. Deletion analysis of the alphaB-crystallin promoter region revealed that both the proximal and the distal heat shock elements were necessary for the transactivation. These results raise the possibility that there is a signal transduction pathway mediating stress signals for the accumulation of alphaB-crystallin by HSF1 and PKN.

Catalysis↗

PubMed1998

Novel, highly potent aldose reductase inhibitors: (R)-(-)-2-(4-bromo-2-fluorobenzyl)-1,2,3,4- tetrahydropyrrolo[1,2-a]pyrazine -4-spiro-3'-pyrrolidine-1,2',3,5'-tetrone (AS-3201) and its congeners.

A series of novel tetrahydropyrrolo[1,2-a]pyrazine derivatives were synthesized and evaluated as aldose reductase inhibitors (ARIs) on the basis of their abilities to inhibit porcine lens aldose reductase (AR) in vitro and to inhibit sorbitol accumulation in the sciatic nerve of streptozotocin-induced diabetic rats in vivo. Of these compounds, spirosuccinimide-fused tetrahydropyrrolo[1, 2-a]pyrazine-1,3-dione derivatives showed significantly potent AR inhibitory activity. In the in vivo activity of these derivatives, 2-(4-bromo-2-fluorobenzyl)-1,2,3,4-tetrahydropyrrolo[1, 2-a]pyrazine-4-spiro-3'-pyrrolidine-1,2',3,5'-tetrone (23t) (SX-3030) showed the best oral activity. The enantiomers of 23t were synthesized, and the biological activities were evaluated. It was found that AR inhibitory activity resides in the (-)-enantiomer 43 (AS-3201), which was 10 times more potent in inhibition of the AR (IC50 = 1.5 x 10(-8) M) and 500 times more potent in the in vivo activity (ED50 = 0.18 mg/kg/day for 5 days) than the corresponding (+)-enantiomer 44 (SX-3202). From these results, AS-3201 was selected as the candidate for clinical development. The absolute configuration of AS-3201 was also established to be (R)-form by single-crystal X-ray analysis. In this article we report the preparation and structure-activity relationship (SAR) of tetrahydropyrrolopyrazine derivatives including a novel ARI, AS-3201.

Aldehyde Reductase↗

PubMed1998

Effect of dual inhibition of 5-alpha-reductase and aromatase on spontaneously developed canine prostatic hypertrophy.

BACKGROUND: Our aim was to assess the effect of dual inhibition of 5-alpha-reductase and aromatase on prostate glands. METHODS: We investigated the morphological changes in the prostate gland and the changes in the hormonal environment after administration of finasteride and arimidex to intact canine specimens. The study consisted of four groups: a 5-alpha-reductase only group (5RI only, n = 5); a 5RI plus aromatase-inhibitor combination group (5RI + ARI combination, n = 5); a BPH control group (n = 3); and a castration control group (n = 3). Finasteride (1 mg/kg/day) and the same dose of arimidex were orally administered for 80 days. RESULTS: In the 5RI group, a significant decrease in the serum dihydrotestosterone (DHT) level was found, and prostatic volume was significantly decreased. However, significant increases in serum testosterone (T) and DHT levels were observed, with a concomitant increase in prostatic volume in the 5RI + ARI combination group. Morphometric analysis showed that histopathological findings in the 5RI + ARI combination group were similar to those in the BPH control group. CONCLUSIONS: Dual inhibition of 5-alpha-reductase and aromatase resulted in a significant increase in prostate volume, accompanied by a 3-10-fold increase in serum testosterone levels and a significant increase in testicular volume.

Anastrozole↗

PubMed1998

Proteolytic activation of PKN by caspase-3 or related protease during apoptosis.

PKN, a fatty acid- and Rho-activated serine/threonine kinase having a catalytic domain highly homologous to protein kinase C (PKC), was cleaved at specific sites in apoptotic Jurkat and U937 cells on Fas ligation and treatment with staurosporin or etoposide, respectively. The cleavage of PKN occurred with a time course similar to that of PKCdelta, a known caspase substrate. This proteolysis was inhibited by a caspase inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde. The cleavage fragments were generated in vitro from PKN by treatment with recombinant caspase-3. Site-directed mutagenesis of specific aspartate residues prevented the appearance of these fragments. These results indicate that PKN is cleaved by caspase-3 or related protease during apoptosis. The major proteolysis took place between the amino-terminal regulatory domain and the carboxyl-terminal catalytic domain, and it generated a constitutively active kinase fragment. The cleavage of PKN may contribute to signal transduction, eventually leading to apoptosis.

Amino Acid Sequence↗

PubMed1998

A protein kinase, PKN, accumulates in Alzheimer neurofibrillary tangles and associated endoplasmic reticulum-derived vesicles and phosphorylates tau protein.

A possible role for a protein kinase, PKN, a fatty acid-activated serine/threonine kinase with a catalytic domain homologous to the protein kinase C family and a direct target for Rho, was investigated in the pathology of Alzheimer's disease (AD) using a sensitive immunocytochemistry on postmortem human brain tissues and a kinase assay for human tau protein. The present study provides evidences by light, electron, and confocal laser microscopy that in control human brains, PKN is enriched in neurons, where the kinase is concentrated in a subset of endoplasmic reticulum (ER) and ER-derived vesicles localized to the apical compartment of juxtanuclear cytoplasm, as well as late endosomes, multivesicular bodies, Golgi bodies, secretary vesicles, and nuclei. In AD-affected neurons, PKN was redistributed to the cortical cytoplasm and neurites and was closely associated with neurofibrillary tangles (NFTs) and their major constituent, abnormally modified tau. PKN was also found in degenerative neurites within senile plaques. In addition, we report that human tau protein is directly phosphorylated by PKN both in vitro and in vivo. Thus, our results suggest a specific role for PKN in NFT formation and neurodegeneration in AD damaged neurons.

Aged↗

PubMed1998

Localization of PKN mRNA in the rat brain.

Distribution of mRNA encoding PKN, a fatty acid and RhoA-activated serine/threonine protein kinase with a catalytic domain highly homologous to that of protein kinase C, was investigated in the rat brain using in situ hybridization histochemistry. PKN mRNA proved to be heterogenously distributed. The highest signals were observed in the cerebellum, in limbic systems such as olfactory bulb, hippocampal formation and limbic cortex, and in regions involved in central autonomic and neuroendocrine functions, such as hypothalamic ventromedial, dorsomedial, lateroanterior and arcuate nuclei, paraventricular hypothalamic nucleus and locus coeruleus. PKN mRNA was also highly expressed in dopaminergic neurons such as the ventral tegmental area and substantia nigra pars compacta, in serotonergic raphe neurons, and in cholinergic neurons such as nucleus diagonal band, nucleus basalis, and lateral dorsal tegmental nucleus. The distribution of PKN mRNA differed from that for PKC isoforms. As the localization of PKN mRNA is heterogeneous, PKN may have a specific role in distinct populations of nerve cells.

Animals↗

PubMed1998

Serum factors and the cellular redox status regulate cellular responsiveness to MHC class II-triggered homotypic B cell adhesion.

Cross-linking the major histocompatibility complex (MHC) class II (MHC-II) by their cognate ligands including mAb induces homotypic cell adhesion. It has been shown that merely surface MHC-II expression is insufficient to induce the response. We found that MHC-II-triggered cell adhesion in human B cell lines was absent when serum was removed from medium. Even in the presence of serum, the response was prevented when cells were treated with the glutathione synthesis inhibitor, buthionine sulfoximine, an irreversible catalase inhibitor, aminotriazole, and H2O2. Flow cytometrical analysis showed that these changes in cellular responsiveness were unlikely to be merely the result of altered surface Ag expression. In addition, the response was independent of the two major lymphocyte adhesion receptors, LFA-1 and intercellular adhesion molecule-1 (ICAM-1). These findings suggest that serum- and redox-sensitive intracellular events regulate cellular responsiveness to MHC-II-triggered LFA-1/ICAM-1-independent cell adhesion independently of differentiation.

B-Lymphocytes↗

PubMed1998

Establishment and characterization of an Epstein-Barr virus-transformed B cell line, KM/C8, from a patient with infantile sialic acid storage disease.

Nakano et al. have recently reported a Japanese case of infantile sialic acid storage disease [C. Nakano, Y. Hirabayashi, K. Ohno, T. Yano, T. Mito, M. Sakurai, Brain Dev., 18 (1996) 153-156]. For further etiological analysis of this disease, we prepared the Epstein-Barr virus (EBV)-transformed cell line (LCL) from the peripheral lymphocytes of this patient and performed initial characterization of the cells. Electron microscopy of the cells showed that the cells contained many vacuoles and swelled lysosomes. Cytochemical staining with sialic acid-specific lectin, Limax flavus agglutinin (LFA), showed strong staining on membranes and subcellular organelles on the patient-derived cells, whereas LCL from a normal person was only weakly stained. The cells from the patient contained 5.5-7.3 nmol/107 cells of free N-acetyl neuraminic acid, whereas three strains of LCLs derived from normal persons contained 1 nmol/107 cells. The culture supernatant of LCL from the patient contained 144 nmol/ml of free N-acetyl neuraminic acid, whereas the LCL culture supernatant from normal persons contained 57-73 nmol/ml of free sialic acid, which was the same or only at a slightly higher level than the fresh medium. In addition, cellular acidic sialidase measured as 4-methylumbelliferyl sialidase was elevated (107 nmol 4-methylumbelliferon released/mg cellular protein/60 min). The EBV-LCL from an ISSD patient is considered to remain as the abnormality of the cell donor.

B-Lymphocytes↗

PubMed1998

Functional defects of a muscle-specific calpain, p94, caused by mutations associated with limb-girdle muscular dystrophy type 2A.

p94 (calpain3), a muscle-specific member of the calpain family, has been shown to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A), a form of autosomal recessive and progressive neuromuscular disorder. To elucidate the molecular mechanism of LGMD2A, we constructed nine p94 missense point mutants found in LGMD2A and analyzed their p94 unique properties. All mutants completely or almost completely lose the proteolytic activity against a potential substrate, fodrin. However, some of the mutants still possess autolytic activity and/or connectin/titin binding ability, indicating these properties are not necessary for the LGMD2A phenotypes. These results provide strong evidence that LGMD2A results from the loss of proteolysis of substrates by p94, suggesting a novel molecular mechanism leading to muscular dystrophies.

Animals↗

PubMed1998

PKN interacts with a paraneoplastic cerebellar degeneration-associated antigen, which is a potential transcription factor.

PKN is a fatty acid-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. PKN has been recently reported to interact with a small GTP-binding protein Rho and cytoskeletal proteins such as neurofilament and alpha-actinin. To identify the new components of the PKN-signaling pathway, the yeast two-hybrid system was employed. Using the amino-terminal regulatory domain of PKN as a bait, cDNA encoding a neural antigen PCD17, which is recognized by characteristic antibodies of patients with paraneoplastic cerebellar degeneration, was isolated from a human brain cDNA library. The interaction between PKN and PCD17 was also determined by the in vitro binding analysis. PCD17 was coimmunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and the amino-terminal region of PCD17. PCD17 was phosphorylated by PKN, and the extent of this phosphorylation was enhanced by addition of 40 microM arachidonic acid. The amino-terminal region of PCD17 could form a homodimer in vitro, and PCD17 fused to the Gal4 DNA binding domain showed the transcriptional transactivation of the chloramphenicol acetyltransferase reporter gene linked to 5 Gal4 binding sites and minimal promoter in rat C6 glioma cells. These results suggest the participation of PCD17 in gene expression and lead to a clue for elucidating the PKN signaling pathway from the cytosol to the nucleus.

Animals↗

PubMed1998

Epstein-Barr-virus-infected human T-cell line with a unique pattern of viral-gene expression.

Epstein-Barr-virus(EBV)-gene expression was analyzed in clonal sub-lines of the human T-cell line MT-2 that are persistently infected by the virus with a positive selection marker. Immunoblot analyses showed the expression of EBV proteins associated with the latent viral cycle, including the EBV nuclear antigen 1 (EBNA1) and the latent membrane protein 1 (LMP1), whereas EBNA2 was not detected. RT-PCR further demonstrated the expression of LMP2A, EBV-encoded small RNA 1 (EBER1), and complementary-strand transcripts from the BamHI-A fragment. EBNA2 mRNA was detected at a very low level. In addition to these latent viral functions, the BZLF1 protein, a transcription factor that activates a cascade of replicative-gene expression in EBV-infected B cells, was detected in these MT-2 clones by immunoblot analysis and immuno-enzymatic staining. Moreover, mRNA from other genes of the early virus-replicative cycle, such as BRLF1, BSMLF1, and BALF2, as well as BZLF1 was detected by Northern-blot analysis. Since sub-lines of the human B-cell lines BJAB and Louckes infected with similar recombinant EBV did not express these early genes, these data suggest that MT-2 cells are relatively permissive for expression of early EBV-replicative functions.

Cell Line↗

PubMed1998

Structure and physiology of calpain, an enigmatic protease.

Calpain is one of the most extensively studied proteinases (1). Although its enzymatic and structural properties have been well characterized, neither the structure-function relationship nor physiological functions are completely understood. In recent years, increasing numbers of molecules showing sequence similarity to calpain have been identified and the concept of a "calpain super family" has become general (2, 3). The term "calpain" originally meant a Ca(2+)-activated, neutral, and intracellular cysteine proteinase, although a proteinase domain similar to that of calpain is a prerequisite for a member of the "calpain super family" (4, 5). The molecular diversity of calpain has attracted interest to its structural and functional transition during evolution. Here we describe the state of current knowledge, progress, and clues to the next phase of calpain research.

Calcium-Binding Proteins↗

PubMed1998

The protein kinase N (PKN) gene PRKCL1/Prkcl1 maps to human chromosome 19p12-p13.1 and mouse chromosome 8 with close linkage to the myodystrophy (myd) mutation.

Protein kinase N (PKN) is a fatty acid- and Rho-activated serine/threonine protein kinase involved in the regulation of cell motility by association with cytoskeletal components such as neurofilament and alpha-actinin. We determined the chromosomal location of the human PKN gene PRKCL1 by fluorescence in situ hybridization and by radiation hybrid mapping. The corresponding mouse gene Prkcl1 was mapped by segregation analysis. We found by FISH that PRKCL1 is localized to chromosome 19p12-p13.1 and, more precisely, by radiation hybrid mapping, about 11 cR from EST WI-6344 in subband 19p12. Prkcl1 maps to mouse chromosome 8 between D8Mit6 and junb. This region of mouse Chr 8 shows a scrambled syntenic conservation to human chromosomes 4q, 8p, and 19p. As the mouse mutation myodystrophy myd has been mapped to the same region, Prkcl1 is a candidate gene for myd.

Animals↗

PubMed1998

Rapid and specific reactive oxygen species generation via NADPH oxidase activation during Fas-mediated apoptosis.

To determine Fas-induced reactive oxygen species (ROS) generation in cells sensitive to Fas-mediated apoptosis, chemiluminescence, a strong indicator of ROS generation, was monitored after stimulation with agonistic anti-Fas. Fas ligation resulted in a rapid and sustained ROS generation. The generation reached a maximum within 5 min and was still observed at least 40 min after the stimulation. No significant ROS generation was observed under conditions where apoptosis was absent. Furthermore, an NADPH oxidase inhibitor, diphenylene iodonium, prevented Fas-mediated ROS generation, suggesting that Fas induces ROS generation via stimulation of the oxidase system.

Apoptosis↗

PubMed1998

Comparative effects of GTPgammaS and insulin on the activation of Rho, phosphatidylinositol 3-kinase, and protein kinase N in rat adipocytes. Relationship to glucose transport.

Electroporation of rat adipocytes with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) elicited sizable insulin-like increases in glucose transport and GLUT4 translocation. Like insulin, GTPgammaS activated membrane phosphatidylinositol (PI) 3-kinase in rat adipocytes, but, unlike insulin, this activation was blocked by Clostridium botulinum C3 transferase, suggesting a requirement for the small G-protein, RhoA. Also suggesting that Rho may operate upstream of PI 3-kinase during GTPgammaS action, the stable overexpression of Rho in 3T3/L1 adipocytes provoked increases in membrane PI 3-kinase activity. As with insulin treatment, GTPgammaS stimulation of glucose transport in rat adipocytes was blocked by C3 transferase, wortmannin, LY294002, and RO 31-8220; accordingly, the activation of glucose transport by GTPgammaS, as well as insulin, appeared to require Rho, PI 3-kinase, and another downstream kinase, e.g. protein kinase C-zeta (PKC-zeta) and/or protein kinase N (PKN). Whereas insulin activated both PKN and PKC-zeta, GTPgammaS activated PKN but not PKC-zeta. In transfection studies in 3T3/L1 cells, stable expression of wild-type Rho and PKN activated glucose transport, and dominant-negative forms of Rho and PKN inhibited insulin-stimulated glucose transport. In transfection studies in rat adipocytes, transient expression of wild-type and constitutive Rho and wild-type PKN provoked increases in the translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane; in contrast, transient expression of dominant-negative forms of Rho and PKN inhibited the effects of both insulin and GTPgammaS on HA-GLUT4 translocation. Our findings suggest that (a) GTPgammaS and insulin activate Rho, PI 3-kinase, and PKN, albeit by different mechanisms; (b) each of these signaling substances appears to be required for, and may contribute to, increases in glucose transport; and (c) PKC-zeta may contribute to increases in glucose transport during insulin, but not GTPgammaS, action.

ADP Ribose Transferases↗

PubMed1998

Molecular cloning and characterization of a novel protein kinase C-interacting protein with structural motifs related to RBCC family proteins.

A novel protein kinase C (PKC)-interacting protein was identified by the yeast two-hybrid screening using the regulatory domain of PKC beta I as a bait. The protein contained several structural motifs such as two putative coiled-coil regions, a RING-finger, a B-box, and a B-box-like motif in the order from NH2- to COOH-terminals. The molecular organization of the protein resembles the structure of the RBCC protein family proteins which usually have a RING-finger, a B-box, and a coiled-coil region. Therefore, the protein identified was designated as RBCK1 (RBCC protein interacting with PKC 1). Northern blot analysis showed that RBCK1 gene is expressed ubiquitously among rat tissues. RBCK1 protein associated with PKC beta I and PKC zeta when coexpressed in cultured mammalian cells. By the polymerase chain reaction-assisted DNA-binding site selection and the electrophoretic mobility shift assay, RBCK1 protein was shown to bind to several DNA fragments containing TGG-rich sequences. When the yeast GAL4 DNA-binding domain fused RBCK1 protein was expressed in COS-7 cells harboring the luciferase gene placed under a synthetic promoter containing GAL4-binding sites, the fusion protein showed enhanced transcriptional activity comparing with the GAL4 DNA-binding domain. These results suggest that RBCK1 protein might be a transcription factor that has a role in the signaling pathway through PKC.

Amino Acid Sequence↗

PubMed1998

Comprehension of famous personal and geographical names in global aphasic subjects.

This experiment compared comprehension of famous personal and geographical names with that of common nouns in three groups of severe aphasic subjects across three modalities. Results revealed that the global aphasic group comprehended famous personal names significantly better than they did common nouns and were better in the reading comprehension modality. Comprehension of famous geographical names in the global aphasic group, however, was as impaired as that of common nouns. CT images suggested that the left occipital lobe and the left posterior inferior temporal gyrus, as well as the right hemisphere, may have relevance to the comprehension of written famous personal names.

Aged↗